Role of Interleukin-12 in Protection against Pulmonary Infection with Methicillin-Resistant Staphylococcus aureus

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is a common pathogen associated with nosocomial pneumonia and is an increasing threat for severe community-acquired pneumonia. We have now investigated the role of interleukin-12 (IL-12) in protective immunity against lung infection with MRSA. The importance of IL-12 in protection from pulmonary MRSA infection was demonstrated by the finding that IL-12p35-deficient mice had a lower survival rate, higher bacterial burdens in lung and spleen, and decreased expression of interferon gamma (IFN-γ) in the lung compared to wild-type mice. These effects were completely reversed by replacement intranasal therapy with recombinant IL-12. Furthermore, exogenous IL-12 treatment of wild-type mice 24 h before pulmonary challenge with a lethal dose of MRSA significantly improved bacterial clearance and resulted in protection from death. The IL-12-treated mice had increased numbers of lung natural killer (NK) cells and neutrophils and higher levels of IFN-γ in the lung and serum compared to untreated mice. The major source of IL-12-driven IFN-γ expression in the lung was the NK cell, and the direct target of pulmonary IFN-γ was the lung macrophage, as shown using mice with a macrophage-specific defect in interferon gamma (IFN-γ) signaling (MIIG mice). Importantly, combination therapy with linezolid and IL-12 following intranasal MRSA infection significantly increased survival compared to that of mice receiving linezolid or IL-12 alone. These results indicate that IL-12-based immunotherapy may hold promise for treatment of MRSA pneumonia.

FIG 1

IL-12p35-deficient mice have increased susceptibility to respiratory MRSA infection. (A) WT and IL-12p35^−/− mice were treated i.n. with either 1 μg of IL-12 or PBS vehicle and then 24 h later were i.n. infected with 1 × 10⁸ CFU of MRSA. Survival was monitored every 12 h for 7 days. Each group contained 10 mice, and the results of two independent experiments were pooled. *, P < 0.05 as determined by log rank test. Bacterial loads (B) and cytokine levels (C) in lung and spleen homogenates and in serum were determined 24 h after MRSA infection. Each bar represents the mean ± standard error of the mean (SEM) of the results from 4 to 19 mice/group. The data were pooled from three independent experiments. Statistical analysis was performed by using the Mann-Whitney U test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 2

IL-12 treatment improves protection against respiratory MRSA infection. WT mice were treated i.n. with either 1 μg of IL-12 or PBS vehicle 24 h before i.n. MRSA infection. (A) Treated mice were challenged i.n. with either 1.5 × 10⁸ (left) or 2.5 × 10⁸ (right) CFU. Survival was monitored every 12 h for 7 days. Each group contained 10 to 15 mice, and the results of three independent experiments were pooled. *, P < 0.05; **, P < 0.01 as determined by a log rank test. (B) Lung and spleen bacterial levels 4 and 24 h after challenge i.n. with 1 × 10⁸ CFU of MRSA. Each symbol represents an individual mouse, and the line indicates the mean. Data were pooled from three independent experiments. Statistical analysis was performed by using the Mann-Whitney U test. ***, P < 0.001.

FIG 3

Effect of IL-12 pretreatment on lung cell levels and cytokine production following MRSA challenge. WT mice were treated i.n. with either 1 μg of IL-12 or PBS vehicle 24 h before i.n. infection with 1 × 10⁸ CFU of MRSA. (A) Four hours and 24 h after infection, lungs were harvested and single cell suspensions were prepared. The cells were stained with fluorescent-conjugated antibodies and analyzed by flow cytometry as described in Materials and Methods. (B) Cytokine levels in lung homogenates and serum were determined by ELISA 4 and 24 h after infection. Each symbol represents an individual mouse, and the line indicates the mean. The data were pooled from three independent experiments. Statistical analysis was performed by using the Mann-Whitney U test. *, P < 0.05; ***, P < 0.001.

FIG 4

Intranasal IL-12 treatment induces IFN-γ production by NK cells. WT mice were treated i.n. with either 1 μg of IL-12 or PBS vehicle 24 h before infection with 1 × 10⁸ CFU of MRSA. Lung cells from uninfected mice and mice infected 24 h earlier were isolated and restimulated in vitro with MRSA for 2 h. They were then stained for surface NK1.1, CD4, and CD8 and for cytoplasmic IFN-γ, followed by flow cytometry analysis. (A) Representative dot plots from each experimental group. (B and C) Percentages (B) and numbers (C) of IFN-γ⁺ NK1.1⁺, IFN-γ⁺ CD4⁺, and IFN-γ⁺ CD8⁺ lung cells. Each symbol represents an individual mouse, and the line indicates the mean. The data were pooled from two independent experiments. Statistical analysis was performed by using the Mann-Whitney U test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 5

The importance of NK cells and IFN-γ in IL-12-mediated protection. Mice were treated i.n. with either 1 μg of IL-12 or PBS vehicle 24 h before infection with 1 × 10⁸ CFU of MRSA. (A) Mice were treated with isotype control MAb or with anti-NK1.1 MAb to deplete NK cells and CFU in lungs, and BALF levels were determined 24 h after MRSA infection. (B) CFU were enumerated in the lungs and spleens of WT, IFN-γ^−/−, and MIIG mice 24 h after infection. Each symbol represents the value for an individual mouse. The horizontal line is the mean for the group. Statistical analysis was performed by using the Mann-Whitney U test. (C) WT and IFN-γ^−/− mice were treated i.n. with either 1 μg of IL-12 or PBS vehicle 24 h before infection with 1.5 × 10⁸ CFU of MRSA. Survival was monitored every 12 h for 7 days. Each group contained 10 to 12 mice, and the results of two independent experiments were pooled. Statistical analysis was performed by using the log rank test.*, P < 0.05; **, P < 0.01.

FIG 6

Combination therapy with IL-12 and linezolid improves survival following MRSA pneumonia. WT and IFN-γ^−/− mice were challenged i.n. with 5 × 10⁸ CFU of MRSA and were then treated i.n. 4 and 24 h later with either PBS vehicle, IL-12 alone, linezolid alone, or a combination of IL-12 and linezolid. Survival of infected mice was monitored every 12 h for 7 days. Each group contained 7 to 10 mice, and the results of two independent experiments were pooled. *, P < 0.05 as determined by a log rank test.